Synthesis of 3-amino-thalidomide and its enantiomers

ABSTRACT

The invention provides new and useful analogs of 3-amino-thalidomide. These analogs include S(−)-3-amino-thalidomide and R(+)-3-amino-thalidomide. The invention also provides processes for making these analogs. Further, the invention provides methods for inhibiting angiogenesis and treating angiogenesis-associated diseases, including cancer and macular degeneration, by administering these compounds.

TECHNICAL FIELD

[0001] The present invention relates to methods and compositions forpreventing unwanted angiogenesis in a human or animal. Moreparticularly, the present invention relates to a method for preventingunwanted angiogenesis, particularly in angiogenesis dependent orassociated diseases, by administration of compounds such as enantiomersof 3-amino-thalidomide.

BACKGROUND OF THE INVENTION

[0002] Angiogenesis is the generation of new blood vessels into a tissueor organ. Under normal physiological conditions, humans and animalsundergo angiogenesis only in very specific, restricted situations. Forexample, angiogenesis is normally observed in wound healing, fetal andembryonal development, and formation of the corpus luteum, endometriumand placenta.

[0003] Angiogenesis is controlled through a highly regulated system ofangiogenic stimulators and inhibitors. The control of angiogenesis hasbeen found to be altered in certain disease states and, in many cases,pathological damage associated with the diseases is related touncontrolled angiogenesis. Both controlled and uncontrolled angiogenesisare thought to proceed in a similar manner. Endothelial cells andpericytes, surrounded by a basement membrane, form capillary bloodvessels. Angiogenesis begins with the erosion of the basement membraneby enzymes released by endothelial cells and leukocytes. Endothelialcells, lining the lumen of blood vessels, then protrude through thebasement membrane. Angiogenic stimulants induce the endothelial cells tomigrate through the eroded basement membrane. The migrating cells form a“sprout” off the parent blood vessel where the endothelial cells undergomitosis and proliferate. The endothelial sprouts merge with each otherto form capillary loops, creating a new blood vessel.

[0004] Persistent, unregulated angiogenesis occurs in many diseasestates, tumor metastases, and abnormal growth by endothelial cells. Thediverse pathological disease states in which unregulated angiogenesis ispresent have been grouped together as angiogenic-dependent orangiogenic-associated diseases.

[0005] One example of a disease mediated by angiogenesis is ocularneovascular disease. This disease is characterized by invasion of newblood vessels into the structures of the eye, such as the retina orcornea. It is the most common cause of blindness and is involved inapproximately twenty eye diseases. In age-related macular degeneration,the associated visual problems are caused by an ingrowth of choroidalcapillaries through defects in Bruch's membrane with proliferation offibrovascular tissue beneath the retinal pigment epithelium. Angiogenicdamage is also associated with diabetic retinopathy, retinopathy ofprematurity, corneal graft rejection, neovascular glaucoma, andretrolental fibroplasia. Other diseases associated with cornealneovascularization include, but are not limited to, epidemickeratoconjunctivitis, Vitamin A deficiency, contact lens overwear,atopic keratitis, superior limbic keratitis, pterygium keratitis sicca,sjogrens disease, acne rosacea, phylectenulosis, syphilis, Mycobacteriainfections, lipid degeneration, chemical burns, bacterial ulcers, fungalulcers, Herpes simplex infection, Herpes zoster infections, protozoaninfections, Kaposi's sarcoma, Mooren's ulcer, Terrien's marginaldegeneration, marginal keratolysis, rheumatoid arthritis, systemiclupus, polyarteritis, trauma, Wegener's sarcoidosis, scleritis,Stevens-Johnson's disease, pemphigoid, and radial keratotomy.

[0006] Diseases associated with retinalchoroidal neovascularizationinclude, but are not limited to, diabetic retinopathy, maculardegeneration, sickle cell anemia, sarcoidosis, syphilis, pseudoxanthomaelasticum, Paget's disease, vein occlusion, artery occlusion, carotidobstructive disease, chronic uveitis/vitritis, Mycobacteria infections,lyme's disease, systemic lupus erythematosis, retinopathy ofprematurity, Eales' disease, Behcet's disease, infections causingretinitis or choroiditis, presumed ocular histoplasmosis, Best'sdisease, myopia, optic pits, Stargardt's disease, pars planitis, chronicretinal detachment, hyperviscosity syndromes, toxoplasmosis, trauma andpost-laser complications. Other eye-related diseases include, but arenot limited to, diseases associated with rubeosis (neovascularization ofthe angle) and diseases caused by the abnormal proliferation offibrovascular or fibrous tissue, including all forms of prolificvitreoretinopathy.

[0007] Another angiogenesis associated disease is rheumatoid arthritis.The blood vessels in the synovial lining of the joints undergoangiogenesis. In addition to forming new vascular networks, theendothelial cells release factors and reactive oxygen species that leadto pannus growth and cartilage destruction. Angiogenesis may also play arole in osteoarthritis. The activation of the chondrocytes byangiogenic-related factors contributes to the destruction of the joint.At a later stage, the angiogenic factors promote new bone growth.Therapeutic intervention that prevents the bone destruction could haltthe progress of the disease and provide relief for persons sufferingwith arthritis.

[0008] Chronic inflammation may also involve pathological angiogenesis.Such diseases as ulcerative colitis and Crohn's disease showhistological changes with the ingrowth of new blood vessels and theinflamed tissues. Bartonelosis, a bacterial infection found in SouthAmerica, can result in a chronic stage that is characterized byproliferation of vascular endothelial cells. Another pathological roleassociated with angiogenesis is found in atherosclerosis. The plaquesformed within the lumen of blood vessels have been shown to haveangiogenic stimulatory activity.

[0009] The hypothesis that tumor growth is angiogenesis-dependent wasfirst proposed in 1971. (Folkman, New Eng. J. Med., 285:1182-86 (1971)).In its simplest terms, this hypothesis states: “Once tumor ‘take’ hasoccurred, every increase in tumor cell population must be preceded by anincrease in new capillaries converging on the tumor.” Tumor ‘take’ iscurrently understood to indicate a prevascular phase of tumor growth inwhich a population of tumor cells occupying a few cubic millimetersvolume, and not exceeding a few million cells, can survive on existinghost microvessels. Expansion of tumor volume beyond this phase requiresthe induction of new capillary blood vessels. For example, pulmonarymicrometastases in the early prevascular phase in mice would beundetectable except by high power microscopy on histological sections.

[0010] Examples of the indirect evidence which support this conceptinclude:

[0011] The growth rate of tumors implanted in subcutaneous transparentchambers in mice is slow and linear before neovascularization, and rapidand nearly exponential after neovascularization. (Algire, et al., J.Nat. Cancer Inst., 6:73-85 (1945)).

[0012] Tumors grown in isolated perfused organs where blood vessels donot proliferate are limited to 1-2 mm³ but expand rapidly to >1000 timesthis volume when they are transplanted to mice and becomeneovascularized. (Folkman, et al., Annals of Surgery, 164:491-502(1966)).

[0013] Tumor growth in the avascular cornea proceeds slowly and at alinear rate, but switches to exponential growth afterneovascularization. (Gimbrone, Jr., et al., J. Nat. Cancer Inst.,52:421-27 (1974)).

[0014] Tumors suspended in the aqueous fluid of the anterior chamber ofthe rabbit eye remain viable, avascular, and limited in size to <1 mm³.Once they are implanted on the iris vascular bed, they becomeneovascularized and grow rapidly, reaching 16,000 times their originalvolume within 2 weeks. (Gimbrone, Jr., et al., J. Exp. Med.,136:261-76).

[0015] When tumors are implanted on the chick embryo chorioallantoicmembrane, they grow slowly during an avascular phase of >72 hours, butdo not exceed a mean diameter of 0.93+0.29 mm. Rapid tumor expansionoccurs within 24 hours after the onset of neovascularization, and by day7 these vascularized tumors reach a mean diameter of 8.0+2.5 mm.(Knighton, British J. Cancer, 35:347-56 (1977)).

[0016] Vascular casts of metastases in the rabbit liver revealheterogeneity in size of the metastases, but show a relatively uniformcut-off point for the size at which vascularization is present. Tumorsare generally avascular up to 1 mm in diameter, but are neovascularizedbeyond that diameter. (Lien, et al., Surgery, 68:334-40 (1970)).

[0017] In transgenic mice which develop carcinomas in the beta cells ofthe pancreatic islets, pre-vascular hyperplastic islets are limited insize to <1 mm. At 6-7 weeks of age, 4-10% of the islets becomeneovascularized, and from these islets arise large vascularized tumorsof more than 1000 times the volume of the pre-vascular islets. (Folkman,et al., Nature, 339:58-61 (1989)).

[0018] A specific antibody against VEGF (vascular endothelial growthfactor) reduces microvessel density and causes “significant or dramatic”inhibition of growth of three human tumors which rely on VEGF as theirsole mediator of angiogenesis (in nude mice). The antibody does notinhibit growth of the tumor cells in vitro. (Kim, et al., Nature,362:841-44 (1993)).

[0019] Anti-bFGF monoclonal antibody causes 70% inhibition of growth ofa mouse tumor which is dependent upon secretion of bFGF as its onlymediator of angiogenesis. The antibody does not inhibit growth of thetumor cells in vitro. (Hori, et al., Cancer Res., 51:6180-84 (1991)).

[0020] Intraperitoneal injection of bFGF enhances growth of a primarytumor and its metastases by stimulating growth of capillary endothelialcells in the tumor. The tumor cells themselves lack receptors for bFGF,and bFGF is not a mitogen for the tumors cells in vitro. (Gross, et al.,Proc. Am. Assoc. Cancer Res., 31:79 (1990)).

[0021] A specific angiogenesis inhibitor (AGM-1470) inhibits tumorgrowth and metastases in vivo, but is much less active in inhibitingtumor cell proliferation in vitro. It inhibits vascular endothelial cellproliferation half-maximally at 4 logs lower concentration than itinhibits tumor cell proliferation. (Ingber, et al., Nature, 48:555-57(1990)). There is also indirect clinical evidence that tumor growth isangiogenesis dependent.

[0022] Human retinoblastomas that are metastatic to the vitreous developinto avascular spheroids which are restricted to less than 1 mm³ despitethe fact that they are viable and incorporate ³H-thymidine (when removedfrom an enucleated eye and analyzed in vitro).

[0023] Carcinoma of the ovary metastasizes to the peritoneal membrane astiny avascular white seeds (1-3 mm³). These implants rarely grow largeruntil one or more of them becomes neovascularized.

[0024] Intensity of neovascularization in breast cancer (Weidner, etal., New Eng. J. Med., 324:1-8 (1991); Weidner, et al., J Nat. CancerInst., 84:1875-87 (1992)) and in prostate cancer (Weidner, et al., Am.J. Pathol., 143(2):401-09 (1993)) correlates highly with risk of futuremetastasis.

[0025] Metastasis from human cutaneous melanoma is rare prior toneovascularization. The onset of neovascularization leads to increasedthickness of the lesion and an increased risk of metastasis.(Srivastava, et al., Am. J. Pathol., 133:419-23 (1988)).

[0026] In bladder cancer, the urinary level of an angiogenic protein,bFGF, is a more sensitive indicator of status and extent of disease thanis cytology. (Nguyen, et al., J. Nat. Cancer Inst., 85:241-42 (1993)).

[0027] Thus, it is clear that angiogenesis plays a major role in themetastasis of cancer. If this angiogenic activity could be repressed oreliminated, then the tumor, although present, would not grow. In thedisease state, prevention of angiogenesis could avert the damage causedby the invasion of the new microvascular system. Therapies directed atcontrol of the angiogenic processes could lead to the abrogation ormitigation of these diseases.

[0028] Angiogenesis has been associated with a number of different typesof cancer, including solid tumors and blood-borne tumors. Solid tumorswith which angiogenesis has been associated include, but are not limitedto, rhabdomyosarcomas, retinoblastoma, Ewing's sarcoma, neuroblastoma,and osteosarcoma. Angiogenesis is also associated with blood-bornetumors, such as leukemias, lymphomas, multiple myeloma, and any ofvarious acute or chronic neoplastic diseases of the bone marrow in whichunrestrained proliferation of white blood cells occurs, usuallyaccompanied by anemia, impaired blood clotting, and enlargement of thelymph nodes, liver and spleen. It is believed to that angiogenesis playsa role in the abnormalities in the bone marrow that give rise toleukemia and lymphoma tumors and multiple myeloma diseases.

[0029] One of the most frequent angiogenic diseases of childhood is thehemangioma. A hemangioma is a tumor composed of newly-formed bloodvessels. In most cases the tumors are benign and regress withoutintervention. In more severe cases, the tumors progress to largecavernous and infiltrative forms and create clinical complications.Systemic forms of hemangiomas, hemangiomatoses, have a high mortalityrate. Therapy-resistant hemangiomas exist that cannot be treated withtherapeutics currently in use.

[0030] Angiogenesis is also responsible for damage found in hereditydiseases such as Osler-Weber-Rendu disease, or heredity hemorrhagictelangiectasia. This is an inherited disease characterized by multiplesmall angiomas, tumors of blood or lymph vessels. The angiomas are foundin the skin and mucous membranes, often accompanied by epitaxis (nosebleeds) or gastrointestinal bleeding and sometimes with pulmonary orhepatitic arteriovenous fistula.

[0031] What is needed, therefore, is a composition and method which caninhibit angiogenesis. What is also needed is a composition and methodwhich can inhibit the unwanted growth of blood vessels, especially intumors.

[0032] Angiogenesis is also involved in normal physiological processes,such as reproduction and wound healing. Angiogenesis is an importantstep in ovulation and also in implantation of the blastula afterfertilization. Prevention of angiogenesis could be used to induceamenorrhea, to block ovulation, or to prevent implantation by theblastula.

[0033] In wound healing, excessive repair or fibroplasia can be adetrimental side effect of surgical procedures and may be caused orexacerbated by angiogenesis. Adhesions are a frequent complication ofsurgery and lead to problems such as small bowel obstruction.

[0034] Several compounds have been used to inhibit angiogenesis. Taylor,et al. (Nature, 297:307 (1982)) have used protamine to inhibitangiogenesis. The toxicity of protamine limits its practical use as atherapeutic. Folkman, et al. (Science, 221:719 (1983), and U.S. Pat.Nos. 5,001,116 and 4,994,443) have disclosed the use of heparin andsteroids to control angiogenesis. Steroids, such as tetrahydrocortisol,which lack gluccocorticoid and mineralocorticoid activity, have beenfound to be angiogenic inhibitors.

[0035] Other factors found endogenously in animals, such as a 4 kDaglycoprotein from bovine vitreous humor and a cartilage derived factor,have been used to inhibit angiogenesis. Cellular factors, such asinterferon, inhibit angiogenesis. For example, interferon alpha or humaninterferon beta have been shown to inhibit tumor-induced angiogenesis inmouse dermis stimulated by human neoplastic cells. Interferon beta isalso a potent inhibitor of angiogenesis induced by allogeneic spleencells. (Sidky, et al., Cancer Res., 47:5155-61(1987)). Human recombinantinterferon (alpha/A) was reported to be successfully used in thetreatment of pulmonary hemangiomatosis, an angiogenesis-induced disease.(White, et al., New Eng. J. Med., 320:1197-1200 (1989)).

[0036] Other agents which have been used to inhibit angiogenesis includeascorbic acid ethers and related compounds. (Japanese Kokai Tokkyo KohoNo.58-13 (1978)). Sulfated polysaccharide DS 4152 also inhibitsangiogenesis. (Japanese Kokai Tokkyo Koho No. 63-119500). Additionalanti-angiogenic compounds include Angiostatin® (U.S. Pat. Nos.5,639,725; 5,792,845; 5,885,795; 5,733,876; 5,776,704; 5,837,682;5,861,372, and 5,854,221) and Endostatin™ (U.S. Pat. No. 5,854,205).

[0037] Another compound which has been shown to inhibit angiogenesis isthalidomide. (D'Amato, et al., Proc. Natl. Acad. Sci., 90:4082-85(1994)). Thalidomide is a hypnosedative that has been successfully usedto treat a number of angiogenesis-associated diseases, such asrheumatoid arthritis (Gutierrez-Rodriguez, Arthritis Rheum., 27(10):1118-21 (1984); Gutierrez-Rodriguez, et al., J. Rheumatol.,16(2):158-63 (1989)), Behcet's disease (Handley, et al, Br. J.Dermatol., 127 Suppl, 40:67-8 (1992); Gunzler, Med. Hypotheses,30(2):105-9 (1989)), graft versus host rejection (Field, et al., Nature,211(55): 1308-10 (1966); Heney, et al., Br. J. Haematol., 78 (1):23-7(1991)), Mycobacteria diseases (Vicente, et al., Arch. Intern. Med.,153(4):534 (1993)), Herpes simplex and Herpes zoster infections (Naafs,et al., Int. J. Dermatol., 24(2):131-4 (1985)), chronic inflammation,ulcerative colitis (Meza, et al., Drug Ther, 23 (11): 74-80, 83 (1993);Powell, et al., Br. J. Dermatol., 113 Suppl 28: 141-4 (1985)), leprosy(Barnes, et al., Infect. Immun., 60(4):1441-46 (1992)) and lupus(Burrows, BMJ, 307: 939-40 (1993)).

[0038] Although thalidomide has minimal side effects in adults, it is apotent teratogen. Thus, there are concerns regarding its use in women ofchild-bearing age. Although minimal, there are a number of side effectswhich limit the desirability of thalidomide as a treatment. One suchside effect is drowsiness. In a number of therapeutic studies, theinitial dosage of thalidomide had to be reduced because patients becamelethargic and had difficulty functioning normally. Another side effectlimiting the use of thalidomide is peripheral neuropathy, in whichindividuals suffer from numbness and disfunction in their extremities.

[0039] Thus, improved methods and compositions are needed that areeasily administered and capable of inhibiting angiogenesis.

SUMMARY OF THE INVENTION

[0040] The present invention provides new derivatives to3-amino-thalidomide, which are analogs of thalidomide. Specifically, thepresent invention provides for the individual R(+) and S(−) enantiomersof 3-amino-thalidomide and processes for preparing these enantiomers.

[0041] The enantiomers of the present invention have the followingstructures:

[0042] In another aspect of the present invention, compositions andmethods are provided that are effective in inhibiting abnormal mitosisand/or unwanted angiogenesis. These compositions are easily administeredby different routes including oral and can be given in dosages that aresafe and provide mitotic and/or angiogenic inhibition at internal sites.The present invention provides a method of treating mammalian diseasesmediated by undesired and uncontrolled mitosis and/or angiogenesis byadministering a composition comprising an anti-mitotic and/oranti-angiogenic compound in a dosage sufficient to inhibit angiogenesis.

[0043] Other features and advantages of the invention will be apparentfrom the following description of preferred embodiments thereof.

[0044] These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and the appended claims.

BRIEF DESCRIPTION OF THE FIGURE

[0045]FIG. 1 illustrates the results of XTT proliferation with HS-Sultancells.

[0046]FIG. 2 illustrates the results of corneal micropocket assaysemploying the compounds of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0047] The present invention is directed to compositions and methods forthe treatment of diseases that are mediated by abnormal mitosis and/orangiogenesis. As described below, compounds employed in the presentinvention exhibit anti-mitotic, anti-angiogenic, and/or anti-tumorproperties. Further, in accordance with the present invention, a methodis provided to synthesize substantially enantiomerically pureS(−)-3-amino-thalidomide and R(+)-3-amino-thalidomide. In accordancewith the present invention, S(−) and R(+) enantiomers of3-amino-thalidomide have anti-mitotic and angiogensis inhibitoryactivities and are useful for the treatment of a number of diseases,including various cancers and macular degeneration.S(−)3-amino-thalidomide showed potent anti-angiogenic and anti-tumoractivity in various in-vito and in-vivo tumor models.

[0048] The enantiomers of the present invention have the followingstructures:

[0049] In one aspect of the present invention, S(−)-3-amino-thalidomideand R(+)-3-amino-thalidomide may be synthesized from S(−) and R(+)enantiomers of 3-nitro-thalidomide, respectively. For example,S(−)-3-amino-thalidomide may be produce in accordance with the followingreaction scheme:

[0050] The S(−)- and R(+) enantiomers of 3-amino-thalidomide can beprovided as pharmaceutically and physiologically acceptable formulationsusing methods and techniques known to those of ordinary skill in theart. These formulations can be administered by standard routes. Ingeneral, the combinations may be administered by the topical,transdermal, oral, rectal or parenteral (e.g., intravenous,subcutaneous, or intramuscular) route. In addition, the combinations maybe incorporated into biodegradable polymers allowing for sustainedrelease of the compound, the polymers being implanted in the vicinity ofwhere drug delivery is desired, for example, at the site of a tumor. Thebiodegradable polymers and their use are described, for example, indetail in Brem et al., J. Neurosurg. 74:441-446 (1991).

[0051] The dosage of the compound will depend on the condition beingtreated, the particular compound, and other clinical factors such asweight and condition of the human or animal and the route ofadministration of the compound. It is to be understood that the presentinvention has application for both human and veterinary use. For oraladministration to humans, a dosage of between approximately 0.1 to 300mg/kg/day, preferably between approximately 0.5 and 50 mg/kg/day, andmost preferably between approximately 0.1 to 2 mg/kg/day, is generallysufficient.

[0052] The formulations include those suitable for oral, rectal, nasal,ophthalmic (including intravitreal or intracameral), topical (includingbuccal and sublingual), vaginal or parenteral (including subcutaneous,intramuscular, intravenous, intradermal, intraocular, intratracheal, andepidural) administration. The formulations may conveniently be presentedin unit dosage form and may be prepared by conventional pharmaceuticaltechniques. Such techniques include the step of bringing intoassociation the active ingredient and the pharmaceutical carrier(s) orexcipient(s). In general, the formulations are prepared by uniformly andintimately bringing into associate the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

[0053] Formulations of the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets, or tablets each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or a suspension in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water-in-oil emulsion and as a bolus, etc.

[0054] A tablet may be made by compression or molding, optionally withone or more accessory ingredients. Compressed tablets may be prepared bycompressing, in a suitable machine, the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surface-active ordispersing agent. Molded tablets may be made by molding, in a suitablemachine, a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may be optionally coated or scored and maybe formulated so as to provide a slow or controlled release of theactive ingredient therein.

[0055] Formulations suitable for topical administration in the mouthinclude lozenges comprising the ingredients in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the ingredient to be administeredin a suitable liquid carrier.

[0056] Formulations suitable for topical administration to the skin maybe presented as ointments, creams, gels and pastes comprising theingredient to be administered in a pharmaceutical acceptable carrier. Apreferred topical delivery system is a transdermal patch containing theingredient to be administered.

[0057] Formulations for rectal administration may be presented as asuppository with a suitable base comprising, for example, cocoa butteror a salicylate.

[0058] Formulations suitable for nasal administration, wherein thecarrier is a solid, include a coarse powder having a particle size, forexample, in the range of 20 to 500 microns which is administered in themanner in which snuff is administered, i.e., by rapid inhalation throughthe nasal passage from a container of the powder held close up to thenose. Suitable formulations, wherein the carrier is a liquid, foradministration, as for example, a nasal spray or as nasal drops, includeaqueous or oily solutions of the active ingredient.

[0059] Formulations suitable for vaginal administration may be presentedas pessaries, tampons, creams, gels, pastes, foams, or sprayformulations containing in addition to the active ingredient suchcarriers as are known in the art to be appropriate.

[0060] Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats, and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example, sealed ampules andvials, and may be stored in a freeze-dried (lyophilized) conditionsrequiring only the addition of the sterile liquid carrier, for example,water for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described.

[0061] Preferred unit dosage formulations are those containing a dailydose or unit, daily sub-dose, as herein above recited, or an appropriatefraction thereof, of the administered ingredient.

[0062] It should be understood that in addition to the ingredients,particularly mentioned above, the formulations of the present inventionmay include other agents conventional in the art having regard to thetype of formulation in question, for example, those suitable for oraladministration may include flavoring agents.

[0063] The present invention can be used to treat diseases characterizedby abnormal cell mitosis. Further, the present invention may be employedto treat any disease characterized by angiogensis. Such diseasesinclude, but are not limited to: abnormal stimulation of endothelialcells (e.g., atherosclerosis), solid tumors and tumor metastasis, benigntumors, for example, hemangiomas, acoustic neuromas, neurofibromas,trachomas, and pyrogenic granulomas, vascular malfunctions, abnormalwound healing, inflammatory and immune disorders, Bechet's disease, goutor gouty arthritis, abnormal angiogenesis accompanying: rheumatoidarthritis, skin diseases, such as psoriasis, diabetic retinopathy, andother ocular angiogenic diseases such as retinopathy of prematurity(retrolental fibroplasic), macular degeneration, corneal graftrejection, neuroscular glaucoma, liver diseases and Oster Webbersyndrome (Osler-Weber Rendu disease).

[0064] Diseases associated with corneal neovascularization that can betreated according to the present invention include but are not limitedto, diabetic retinopathy, retinopathy of prematurity, corneal graftrejection, neovascular glaucoma and retrolental ibroplasias, epidemickeratoconjunctivitis, Vitamin A deficiency, contact lens overwear,atopic keratitis, superior limbic keratitis, pterygium keratitis sicca,sjogrens, acne rosacea, phylectenulosis, syphilis, Mycobacteriainfections, lipid degeneration, chemical burns, bacterial ulcers, fungalulcers, Herpes simplex infections, Herpes zoster infections, protozoaninfections, Kaposi's sarcoma, Mooren's ulcer, Terrien's marginaldegeneration, mariginal keratolysis, trauma, rheumatoid arthritis,systemic lupus, polyarteritis, Wegener's sarcoidosis, scleritis,Steven-Johnson disease, pemphigoid radial keratotomy, and corneal graphrejection.

[0065] Diseases associated with retinal/choroidal neovascularizationthat can be treated according to the present invention include, but arenot limited to, diabetic retinopathy, macular degeneration, sickle cellanemia, sarcoid, syphilis, pseudoxanthoma elasticum, Paget's disease,vein occlusion, artery occlusion, carotid obstructive disease, chronicuveitis/vitritis, mycobacterial infections, Lyme's disease, systemiclupus erythematosis, retinopathy of prematurity, Eales' disease,Behcet's disease, infections causing a retinitis or choroiditis,presumed ocular histoplasmosis, Best's disease, myopia, optic pits,Stargart's disease, pars planitis, chronic retinal detachment,hyperviscosity syndromes, toxoplasmosis, trauma and post-lasercomplications. Other diseases include, but are not limited to, diseasesassociated with rubeosis (neovasculariation of the angle) and diseasescaused by the abnormal proliferation of fibrovascular or fibrous tissueincluding all forms of proliferative vitreoretinopathy, whether or notassociated with diabetes.

[0066] Another disease which can be treated according to the presentinvention is rheumatoid arthritis. It is believed that the blood vesselsin the synovial lining of the joints undergo angiogenesis. In additionto forming new vascular networks, the endothelial cells release factorsand reactive oxygen species that lead to pannus growth and cartilagedestruction. The factors involved in angiogenesis may activelycontribute to, and help maintain, the chronically inflamed state ofrheumatoid arthritis.

[0067] Another disease that can be treated according to the presentinvention are hemangiomas, Osler-Weber-Rendu disease, or hereditaryhemorrhagic telangiectasia, solid or blood borne tumors and acquiredimmune deficiency syndrome.

[0068] Studies of the S(−)- and R(+) enantiomers of 3-amino-thalidomide,particularly S(−)-3-amino-thalidomide, show that these compounds are aspotent as inhibitors of angiogenesis. These studies indicate that thesecompounds are useful for the treatment of angiogensesis-associateddiseases. As indicated above, one angiogenesis-associated group ofdiseases is cancer. Numerous tumors, including solid tumors andblood-borne tumors, require angiogenesis to grow beyond a very smallsize. Inhibition of angiogenesis will result in inhibition of growth ofthe tumor. Examples of specific types of cancer which can be treatedwith the S(−)- and R(+) enantiomers of 3-amino-thalidomide include, butare not limited to, prostate cancer, breast cancer, cervical cancer,uterine cancer, ovarian cancer, gliomas, hemangiomas, Kaposi's sarcoma,pancreatic cancer, retinoblastomas, melanomas, bladder cancer,rhabdomyosarcomas, retinoblastomas, Ewing's sarcoma, neuroblastomas,osteosarcoma, leukemias, lymphomas, multiple myeloma, and various acuteand chronic neoplastic diseases of the bone marrow. The S(−)- and R(+)enantiomers of 3-amino-thalidomide also inhibit metastases of existingtumors. Examples of metastases which can be inhibited include, but arenot limited to, bone metastases, lung metastases, liver metastases, andperitoneal metastases.

[0069] This invention is further illustrated by the following examples,which are not to be construed in any way as imposing limitations uponthe scope thereof On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims. The table below provides desired embodiments, while the Examplesprovide the synthesis of representative compounds.

EXAMPLES

[0070] The following compounds were synthesized by modification inmethods described in Shealy, et al. J. Pharm. Sci., 1968, 57, 757-764;Polonski, et al. J. Chem. Soc. Perkin Trans. I, 1988, 639-648; Muller,et al. Bioorg. Med. Chem. Lett., 1999, 9, 1625-1630; Helm, et al.Arzneim-Forsch./Drug Res., 1981, 31, 941-949; Shah, et al., J. Med.Chem., 1999, 42, 3014-3017; and Menard, et al., Can. J. Che., 1963, 41,1722-1725.

Example 1 Synthesis of S(−)-(3-benzyloxycarbonylamino)-glutamide

[0071] Into a stirring solution of carboxybenzyloxy-L-glutamine (2.8 g,10 mmols) in 40 mL anhydrous THF (tetrahydrofuiran),1,1-carbonyldiimidazole (1.92 g, 12 mmols) were added. The reactionmixture was heated under reflux for 18 hours. The THF was evaporated andthe product was dissolved in chloroform. The chloroform layer was washedwith water and brine and dried over anhydrous CaSO₄, filtered andevaporated to give white solid. The solid product was crystallized fromethyl ether to give 2.4 grams crystalline powder (90%). (Alternatively,carboxybenzyloxy-L-glutamine can be cyclized by treating with SOCl₂ inDMF (N,N-dimethylformamide) at −70° C. to 0° C. for 1 hour to formS(−)-(3-benzyloxycarbonylamino)-glutarimide. The reaction mixture wasdiluted with CHCl₃ and washed with 5% Na₂CO₃, dried over anhydrousNa₂SO₄, filtered, and evaporated to give 2.5 g (90% yield)S(−)-(3-benzyloxycarbonylamino)-glutarimide).

[0072] 1H NMR in CDCl₃ confirmed the product asS(−)-(3-benzyloxycarbonylamino)-glutarimide). 1H NMR (CDCL₃, PPM), 8.2(1H, s broad), 7.4 (5H, s, aromatic), 5.8 (1H, d), 5.15 (2H, s), 4.4(1H, dd, J=4.5, 3), 2.95-2.4 (3H, m), 1.86 (1H, d, t, J=11.5, 6.5). m.p. 122-124° C. (lit=122-124° C.).

Example 2 Synthesis of S(−)-3-Amino-glutarimide.HBr

[0073] Into a solution of S-(−)-(3-benzyloxycarbonylamino)-glutarimide(1.2 g, 4.6 mmols) in 15 mL acetic acid glacial, 8 mL of 30% HBr/aceticacid solution was added at 20° C. The temperature of reaction mixturewas raised to RT and stirred for 1 hour. White solid powder ofS-(−)-2-Amino-gluteramide.HBr started appearing in reaction mixture. Thesolid was filtered and washed with 5 mL acetic acid glacial and thenwith ether to give 1.8 g (80%) product. Analysis on polarimeter ofproduct showed (−) rotation, [a]25D (c=1, water)=−37.5 o and confirmedthe product as S(−)-2-amino-gluteramide. 1H NMR in DMSO-D6 confirmed theproduct as 2-amino-L-gluteramide.HBr. 1H NMR (DMSO-D6, PPM).

Example 3 Synthesis of S(−)-3-Nitro-thalidonide

[0074] Into a solution of (4.18 g, 20 mmols) 3-amino-gluteramide-HBr in50 mL of anhydrous DMF, 3.8g (20 mmols) of 3-nitrophthalic anhydride wasadded. After adding 100 mL acetic acid (glacial), the reaction mixturewas heated at about 70° C. to about 80° C. for about 24 hours.Thereafter, the solvents were evaporated under vacuum to yield anoff-white solid. On adding 10 mL ethyl alcohol to the solid, anoff-white powder product was formed. The product was separated andwashed with 20 mL ethyl alcohol.

[0075] 1H NMR in DMSO-D6 confirmed the product asS(−)-3-nitro-tholidomide. m. p. 228-229° C. (lit=228.5-229.5° C.). 1HNMR (DMSO-D₆, PPM),11.25 (1H, s broad), 8.35 (1H, d, J=7.2), 8.25 (1H,d, J=7.0), 8.15 (1H, t, J=8.0), 5.2 (1H, dd, J=5.5, 7.2), 3.00-2.85 (1H,m), 2.65-2.4 (2H, m), 2.15-2.05 (1H, m).

Example 4 Synthesis of S(−)-3-Amino-thalidomide

[0076] 3-nitro-thalidomide (1 g, 3.3 mmols) was dissolved in 50 mLdioxane/methanol 4:1 mixture and hydrogenated in a Parr hydrogenater at40 psi of hydrogen in the presence of a Pd/C 5% catalyst for about 4hours. After filtering the reaction mixture through a Celite filteringagent, the solvents were evaporated under vacuum to yield a yellowpowder. The product was recrystallized from ethyl acetate/dioxane toyield 800 mg (85% purity) of S(−)-3-amino-thalidomide.

[0077] 1H NMR in DMSO-D₆ confirmed the product asS(−)-3-amino-thalidomide. m. p. 318.2-319.5° C. 1H NMR (DMSO-D₆, PPM),11.10 (1H, s broad), 7.45 (1H, t, J=7.5), 7.05 (1H, d, J=5.2), 6.95 (1H,d, J=5.2), 6.5 (2H, s broad), 5.05 (1H, dd, J=5.0, 13.42), 2.95-2.80(1H, m), 2.65-2.5 (2H, m), 2.05-1.95 (1H, m). Absolute configuration wasdetermined by comparison of specific rotation [a]²⁵ _(D) of R- andS-3-amino-thalidomide to the analogous compounds R(+)- andS(−)-thalidomide. Analysis on polarimeter of product showed (−)rotation, [a]²⁵ _(D) (C=0.5, dioxane)=−27.7.0° and confirmed the productas S(−)-3-amino-thalidomide.

[0078] The two enantiomers of 3-amino-thalidomide were resolved bychiral HPLC column Welk-01 (10 mm×750 mm) and eluted with CH3CN/MeOH/H2O1:1:5 mixture. The retention time for the S(−) enantiomer was 33.74minutes and for the R (+) enantiomer 35.62 minutes at a flow rate of2mL/min at 240 nm, respectively.

Example 5 Synthesis of R(+)-3-amino-thalidomide

[0079] Compound R-(+)-3-amino-thalidomide was synthesized by the sameprocedure as for S-(−)-3-amino-thalidomide above, except the synthesiswas started with commercially available carboxybenzyloxy-D-glutamine,which formed R(+)-3-nitro-thalidomide (See Example 1). Analysis onpolarimeter of product showed (+) rotation [a]²⁵ _(D) (c=1,dioxanesl)=+37.0° and confirmed the product as R(+)-3-amino-thalidomide.1H NMR in DMSO-D₆ confirmed the product as 3-amino-thalidomide.

Example 6 Synthesis of S(−)-3-Amino-thalidomide (constructive example)

[0080] S(−)-3-amino-thalidomide may be synthesized by dissolvingS(−)-3-nitro-thalidomide in concentrated HCl and then treating thereaction mixture with granulateasd tin. After heating the reactionmixture at about 70° C. to about 80° C. for about 2 hours, the reactionmixture should be filtered and the acid evaporated under vacuum to yielda yellow powder. The product should be recryrstallized from water andthen ethyl acetate/dioxane to yield S(−)-3-amino-thalidomide.

Example 7

[0081] The Roche Cell Proliferation Kit II (XTT) is an useful assay forscreening the relative efficacy of small molecules. The assayquantitatively determines cellular proliferation in response to agonistsand/or antagonists of proliferation. It is based on the cleavage of theyellow tetrazolium salt (XTT) by metabolically active/viable cells toform an orange formazan dye. The formation of the soluble dye allowsdirect quantification using a scanning mulitwell spectrophotometer. Anincrease in the number of living cells (resulting from proliferation)results in a greater production of formazan dye which corresponds to anincrease in the absorbance value.

[0082] When evaluating analogs of thalidomide, or the like, we haveemployed HS-Sultan cells in an in vitro XTT assay. In each well of a96-well microtiter plate, cells are seeded at a density of 15,000 cellsper 90 μL of normal growth media approximately 16 hours prior totreatments. During culture and treatments, cells are maintained at 37°C. with 5% CO₂ in a high humidity incubator. Treatments (10×) are addedin 10 μL aliquots to achieve a 1× final treatment concentration in eachwell. Each concentration is done in triplicate. The XTT labeling mixtureis added in 50 μL aliquots to each well during the final four hours ofthe 72 hour treatment period. When the treatment/labeling period iscomplete, the plate is read on a spectrophotometric plate reader at awavelength of 470 nm and a 650 nm reference wavelength. For individualexperiments, the average absorbance values (with background subtracted)for each treatment are plotted against the concentration. A largerabsorbance value corresponds to a greater amount of proliferation. Anegative control (untreated cells) is used as a point of reference; anabsorbance value less than the control reflects inhibition ofproliferation.

[0083] When comparing experiments conducted over a period of time,absorbance values from each experiment may vary due to a number offactors (degradation of the XTT reagents over time is the most commonfactor). When using reagents from an older XTT kit or switching to a newkit, the overall absorbance values for that individual experiment may behigher or lower, making a direct comparison to another experimentdifficult. Therefore, it is common practice to convert the absorbancevalues to a ratio of the treated values divided by the negative controlvalue (treatment over control) when comparing the results from multipleexperiments; the “treatment over control” values for each treatment arethen plotted against the μM concentration. FIG. 1 compares the3-amino-thalidomide enantiomers. The R and the R,S plots represent datapooled from 3 experiments. The S and thalidomide plots represent datapooled from about 12 experiments. As illustrated in FIG. 1, both theS(−)- and the R(+)-3-amino-thalidomide enantiomers show anti-cellularproliferation activity.

Example 8

[0084] Antitumor activity has been evaluated for the S(−) and the R(+)3-amino-thalidomide enantiomers as follows. For the HsS 5 cell line, 2million cells are injected into the tail vein of 8 week old, female,SCID mice. Treatment is initiated after two weeks and continued dailyuntil the mice die or show hind limb paralysis. Results are expressed asmean time to death in treated versus control animals. For the Lewis lungmodel, 2.5×10(5) cells are injected intravenously into the tail vein of6-8 week old, male, C57BL/6 mice and treatment is initiated on daythree. The time period of treatment is usually 11-15 days in duration.After sacrifice by CO₂ asphyxiation, lungs (where tumors seed and grow)are removed, rinsed in water, patted dry, and weighed. Mean lung weightsof age-matched, non-tumor bearing mice are subtracted from the weight oftreated, tumor-bearing mice with results expressed as lung weight gainin treated versus control animals. Table 1 summarizes data from in vivoexperiments in lung and plasma cell tumor metastatic tumor systems,comparing the antitumor activity of the three enantiomeric preparationsof 3-amino thalidomide. These data demonstrate that that the S(−)enantiomer was the most active enantiomer of 3-amino thalidomide in eachtumor model. TABLE 1 Dose Tumor Model Test Agent mg/kg/day ActivityHsSultan B-cell lymphoma Vehicle — 26 days* metastatic in SCID mice(0.5% methyl cellulose) R, S  50 41 days* R(+)  50 37 days* S(−)  50 47days* HsSultan B-cell lymphoma Vehicle — 26 days* metastatic in SCIDmice (0.5% methyl cellulose) R, S 200 28 days* R(+) 200 31 days* S(−)200 47 days* Lewis lung carcinoma Vehicle — 0.40 g # metastatic inC57BL/6 mice (0.5% methyl cellulose) R, S 100 0.27 g # R(+) 100 0.42 g #S(−) 100 0.17 g #

Example 9

[0085] A corneal micropocket assay was performed as described in Kenyon,et al., A Model of Angiogenesis in the Mouse Cornea, Invest. Ophtalmol.& Vis. Sci., 37, 1625-1632 (1996), which is incorporated herein in itsentirety. Pellets were used containing 80 ng of bFGF or human recombinatVEGF (R&D Systems, Minneapolis, Minn.) in C57BL/6J mice. The treatedgroups received daily administration for five (bFGF) or six (VEGFconsecutive days of thalidomide, S(−)- and R(+)-3-amino-thalidomide(3APG), S(−)-3APG, and R(+)-3APG (50 mg/kg) suspended in 0.5%carboxymethylcellulose i.p. Treatment was started on the day of pelletimplantation; control mice received only carboxymethylcellulose i.p. Thearea of vascular response was assessed on the fifth (bFGF) or sixth(VEGF) postoperative day using a slit lamp. The results are reported inFIG. 2. As indicated in FIG. 2, differences in bFGF inducedneovascularization between S(−)-3APG and control were significant (n=9each, P<0.0001) as were differences between S(−)-3APG and thalidomide(n=9 each, P<0.01). Differences in VEGF induced neovascularizationbetween S(−)-3APG and control were significant (n=9 each, P<0.001), aswere differences between S(−)-3APG and thalidomide (n=9 each, P<0.01).

[0086] By “an effective amount” is meant a therapeutically orprophylactically effective amount. Such amounts can be readilydetermined by an appropriately skilled person, taking into account thecondition to be treated, the route of administration and other relevantfactors. Such a person will readily be able to determine a suitabledose, mode and frequency of administration.

[0087] It should be understood, of course, tha the foregoing relatesonly to preferred embodiments of the present invention and that numerousmodifications or alterations may be made therein without departing fromthe spirit and the scope of the invention as set forth in the appendedclaims.

What is claimed is:
 1. A pharmaceutical composition comprising apharmaceutically acceptable carrier and a stereoisomer of3-amino-thalidomide, wherein the stereoisomer consists essentially ofS(−)-3-amino-thalidomide or R(+)-3-amino-thalidomide.
 2. The compositionof claim 1, wherein the composition is in the form of tablets, pills,capsules, suppositories, sachets, granules, powders, creams, lotions,ointments, patches, liquid solutions, suspensions, dispersions,emulsions, syurups, liposomes, microparticles, and microcapsules.
 3. Amethod of inhibiting undesired angiogenesis in a human or animalcomprising administering to the human or animal with undesiredangiogenesis an angiogenesis inhibiting amount of 3-amino-thalidomide,wherein the stereoisomer consists essentially ofS(−)-3-amino-thalidomide or R(+)-3-amino-thalidomide.
 4. The method ofclaim 3, wherein the administration is oral, parenteral, rectal,vaginal, topical, transdermal, intravenous, intramuscular,intraperatoneal, or subcutaneous.
 5. The method of claim 3, wherein theeffective amount is from about 0.1 to about 300 mg/kg/day.
 6. The methodof claim 3, wherein the effective amount is from about 0.5 and about 50mg/kg/day.
 7. The method of claim 3, wherein the effective amount isfrom about 0.1 and about 2 mg/kg/day.
 8. The method of claim 3, whereinthe undesired angiogenesis occurs in a disease selected from diabeticretinopathy, retinopathy of permaturity, corneal graph rejection,neovascular glacoma, retrolental fibroplasia, epidemickeratoconjunctivitis, Vitamin A deficiency, contact lens overwear,atopic keratitis, superior limbic keratitis, pterygium keratitis sicca,Sjorgren's syndrom, acne rosacea, phylectenulosis, syphilis,Mycobacteria infections, lipid degeneration, chemical bums, bacterialulcers, fungal ulcers, Herpes simplex infections, Herpes zosterinfections, protozoan infections, Kaposi's sarcoma, Mooren's ulcer,Terrien's marginal degeneration, mariginal keratolysis, trauma,rheumatoid arthritis, systemic lupus, polyarteritis, Wegener'ssarcoidosis, scleritis, Steven-Johnson disease, radial keratotomy,corneal graph rejection, diabetic retinopathy, macular degeneration,sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Paget'sdisease, vein occlusion, artery occlusion, carotid obstructive disease,chronic uveitis/vitritis, Lyme disease, systemic lupus erythematosis,retinopathy of prematurity, Eales' disease, Behcet's disease, infectionscausing a retinitis or choroiditis, presumed ocular histoplasmosis,Best's disease, myopia, optic pits, Stargart's disease, pars planitis,chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis,trauma, post-laser complications, rubeosis, abnormal proliferation offibrovascular or fibrous tissue, proliferative vitreoretinopathy,atherosclerosis, solid tumors, blood borne tumors, tumor metastasis,Bartonellosis, hemangiomas, acoustic neuromas, neurofibromas, trachomas,pyrogenic granulomas, vascular malfunctions, abnormal wound healing,inflammatory and immune disorders, Bechetfs disease, gout, goutyarthritis, rheumatoid arthritis, psoriasis, diabetic retinopathy,retinopathy of prematurity, macular degeneration, neuroscular glaucoma,liver diseases, Osler-Weber Rendu disease, hereditary hemorrhagictelangiectasia, acquired immune deficiency syndrome, rheumatoidarthritis, prostate cancer, breast cancer, cervical cancer, uterinecancer, ovarian cancer, gliomas, hemangiomas, pancreatic cancer,retinoblastomas, melanomas, bladder cancer, rhabdomyosarcomas,retinoblastomas, Ewing's sarcoma, neuroblastomas, osteosarcoma,leukemia, lymphoma, multiple myeloma, or various acute and chronicneoplastic diseases of the bone marrow.
 9. The method of claim 8,wherein the administration is oral, parenteral, rectal, vaginal,topical, transdermal, intravenous, intramuscular, intraperatoneal, orsubcutaneous.
 10. The method of claim 8, wherein the effective amount isfrom about 0.1 to about 300 mg/kg/day.
 11. A method of treating cancerin a human or animal comprising administering to the human or animalhaving cancer a cancer treatment effective amount of a stereoisomer of3-amino-thalidomide, wherein the stereoisomer consists essentially ofS(−)-3-amino-thalidomide or R(+)-3-amino-thalidomide.
 12. The method ofclaim 11, wherein the administration is oral, parenteral, rectal,vaginal, topical, transdermal, intravenous, intramuscular,intraperatoneal, or subcutaneous.
 13. The method of claim 11, whereinthe effective amount is from about 0.1 to about 300 mg/kg/day.
 14. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a stereoisomer of 3-amino-thalidomide, wherein thestereoisomer consists essentially of S(−)-3-amino-thalidomide.
 15. Themethod of claim 14, wherein the administration is oral, parenteral,rectal, vaginal, topical, transdermal, intravenous, intramuscular,intraperatoneal, or subcutaneous.
 16. The method of claim 14, whereinthe effective amount is from about 0.1 to about 300 mg/kg/day.